Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:826-832
Published online before print January 25, 2007, doi: 10.1161/01.ATV.0000258795.75121.8a
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
27/4/826    most recent
01.ATV.0000258795.75121.8av1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Apostolov, E. O.
Right arrow Articles by Basnakian, A. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Apostolov, E. O.
Right arrow Articles by Basnakian, A. G.
(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:826.)
© 2007 American Heart Association, Inc.

Vascular Biology

Carbamylated Low-Density Lipoprotein Induces Monocyte Adhesion to Endothelial Cells Through Intercellular Adhesion Molecule-1 and Vascular Cell Adhesion Molecule-1

Eugene O. Apostolov; Sudhir V. Shah; Ercan Ok; Alexei G. Basnakian

From the Division of Nephrology (E.O.A., S.V.S., E.O., A.G.B.), Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Ark; Renal Medicine Service (S.V.S., A.G.B.), Central Arkansas Veterans Healthcare System, Little Rock, Ark; Division of Nephrology (E.O.), Department of Internal Medicine, Ege University Medical School, Izmir, Turkey.

Correspondence to Alexei G. Basnakian, Division of Nephrology, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St, Slot 501, Little Rock, AR 72205. E-mail basnakianalexeig{at}uams.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Objective— Carbamylated low-density lipoprotein (LDL), the most abundant modified LDL isoform in human blood, has been recently implicated in causing the atherosclerosis-prone injuries to endothelial cells in vitro and atherosclerosis in humans. This study was aimed at testing the hypothesis that carbamylated LDL acts via inducing monocyte adhesion to endothelial cells and determining the adhesion molecules responsible for the recruitment of monocytes.

Methods and Results— Exposure of human coronary artery endothelial cells with carbamylated LDL but not native LDL caused U937 monocyte adhesion and the induction of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 adhesion molecules as measured by cell enzyme-linked immunosorbent assay. Silencing of intercellular adhesion molecule-1 by siRNA or its inhibition using neutralizing antibody resulted in decreased monocyte adhesion to the endothelial cells. Similar silencing or neutralizing of vascular cell adhesion molecule-1 alone did not have an effect but was shown to contribute to intercellular adhesion molecule-1 when tested simultaneously.

Conclusions— Taken together, these data provide evidence that intercellular adhesion molecule-1 in cooperation with vascular cell adhesion molecule-1 are essential for monocyte adhesion by carbamylated low-density lipoprotein-activated human vascular endothelial cells in vitro.

The exposure of human coronary artery endothelial cells with carbamylated LDL but not native LDL caused U937 monocyte adhesion and the induction of ICAM-1 and VCAM-1 adhesion molecules. Silencing of ICAM-1 and VCAM-1 by siRNA or their inhibition using neutralizing antibody resulted in decreased monocyte adhesion to the endothelial cells.


Key Words: atherosclerosis • carbamylated low-density lipoprotein • endothelial cells • intercellular adhesion molecule-1 • monocyte adhesion • vascular cell adhesion molecule-1


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Monocyte (leukocyte) adhesion to activated vascular endothelial cells and their migration into the vessel wall is the critical event in the initiation of atherosclerosis. This process is caused by the upregulation of adhesion molecules on endothelial cells and an increased expression in the vascular wall of chemotactic factors to monocytes. Highly specific adhesive interactions between monocytes and endothelial cells are mediated by 3 main families of receptors: members of the immunoglobulin superfamily, selectins, and integrins. Recent studies indicate that intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), members of the immunoglobulin superfamily, are the most common participants in monocyte attraction induced by different stimuli.1–3 The expression of adhesion molecules and monocyte adhesion can be triggered by a variety of plasma components, such as interferon-{gamma},4 homocysteine,5 and lipoproteins, like oxidized low-density lipoprotein (oxLDL),6 or its derivates, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine7 and lysophosphatidylcholine.8 The identification of new plasma components that are responsible for monocyte adhesion is important in light of their potential application as pathogenic atherosclerosis markers, predictors or therapeutic targets.

Carbamylated low-density lipoprotein (cLDL) is a recently identified type of modified low-density lipoprotein (LDL) that seems to be important in atherosclerosis in humans.9–11 It is generated by irreversible chemical modification of the protein component of the LDL particle, apolipoprotein B, and by urea-derived cyanate present in human blood plasma.9 Plasma levels of cLDL as determined by sandwich enzyme-linked immunosorbent assay (ELISA) methods vary in a higher range of concentrations than oxLDL, which makes it the most abundant LDL isoform of human plasma.11 The elevation of blood urea in uremic patients causes a proportional increase of plasma cLDL.11 The LDL of chronic renal failure patients on dialysis was shown to induce greater monocyte–endothelial cell adhesion.12 A higher level of plasma cLDL correlates with atherosclerosis in uremic patients.13 Our recent study showed that cLDL induced a variety of damaging stimuli to endothelial and vascular smooth muscle cells, all of which are attributed to atherosclerosis.10,14 These include incorporation by endothelial cells, cytotoxicity toward endothelial cells, and the induction of vascular smooth muscle cell proliferation.

The present study was undertaken to determine whether monocyte adhesion to endothelial cells can be induced by cLDL and to identify the adhesion molecules involved in this process. This in vitro study utilizes U937 monocyte cells and human coronary artery endothelial cells (HCAECs), commonly used for studies of monocyte adhesion.3,10 We demonstrated that cLDL induces ICAM-1 and VCAM-1 on endothelial cells, and the role of these adhesion molecules in the attraction of monocytes in vitro. Inactivation of these pathways by RNA interference or inhibiting the antibody strongly protected against the adhesion of monocytes to endothelial cells, thus providing evidence for the potential importance of these mechanisms in cLDL-mediated atherosclerosis.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
An expanded materials and methods section is available online (please see http://atvb.ahajournals.org).

Native, cLDL, and oxLDLs
Native human LDL (nLDL) and all chemicals were purchased from Sigma (St. Louis, Mo) unless designated otherwise. The cLDL was prepared as previously described by us.10,11 The oxLDL was prepared as described by Kume et al15 and used as a positive control.

Monocyte Adhesion
The U937 cells were chosen because they have the phenotype of the monocytes and are commonly used in monocyte adhesion studies.3,5,6 Monocyte adhesion was determined as described by Koga et al.16

Monocyte adhesion to cLDL-activated HCAECs under flow conditions was performed similarly to methods described elsewhere.17

Cell ELISA
Cell ELISA was performed as described by Frahm et al.18 Antibody titers and optimal reaction conditions were elaborated before the experiment.

Immunocytochemistry
Immunocytochemical staining of LDL-treated cells with polyclonal anti–ICAM-1 (1:100) or anti–VCAM-1 (1:100) antibody (Santa Cruz Biotechnology) was performed similarly as that described by Langer et al.19

ICAM-1 and VCAM-1 siRNA Silencing
For the study of protection from monocyte adhesion, HCAECs were transfected with siRNA to ICAM-1, VCAM-1, or both for 48 hours, then the transfection medium was removed, and the cells were treated with cLDL or vehicle in serum-free medium for 24 hours.

RNA Extraction and Real-Time Reverse-Transcription Polymerase Chain Reaction
The total RNA was extracted using a RNeasy Mini kit from Qiagen (Valencia, Calif) as suggested by the manufacturer. The reverse-transcription reaction was performed using the GeneAmp Gold RNA PCR core kit (Applied Biosystems, Foster City, Calif) using Oligo d(T)16. The reaction mix was prepared using Platinum SYBR Green qPCR Supermix-UDG (Invitrogen Corporation, Carlsbad, Calif) according to manufacturer recommendations.

Statistical Analysis
The results were expressed as mean±SEM. The statistical analysis was performed using SPSS software (SPSS Inc, Chicago, Ill). To evaluate the significance of differences between the 2 groups of experiments, the ANOVA and Student t test were used. Additionally, to evaluate the significance of several time points in comparison to one control point, the Bonferroni adjustment of the t test was used. A value of P<0.05 was considered statistically significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Acceleration of Monocyte Adhesion to Endothelial Cells by cLDL
To determine whether cLDL causes monocyte adhesion to endothelial cells, cLDL (200 µg/mL) was applied to the endothelial cells for different periods of time and BCECF-AM–labeled U937 cells were allowed to adhere for 30 minutes. We found a significant increase of monocyte adhesion to the endothelial cells treated for 12 hours or longer with cLDL, whereas nLDL and the vehicle control did not cause any monocyte adhesion (Figure 1). The oxLDL that was previously described to induce monocyte adhesion,6,20,21 was used as a positive control. A 6-hour or longer treatment with oxLDL (200 µg/mL) caused a 4-fold increase of monocyte adhesion to the endothelial cells. It is interesting that once monocyte adhesion to oxLDL-treated endothelial cells reached the maximum, no further increase of adhesion was observed. Contrary to oxLDL, cLDL caused significant monocyte adhesion versus nLDL and vehicle control only after a 12-hour course, followed then by a further increase of monocyte adhesion over the level of oxLDL. At the end of the 24-hour course, the adhesion induced by cLDL was higher than the one induced by oxLDL.


Figure 1
View larger version (65K):
[in this window]
[in a new window]

  		Figure 1. Time course of the monocyte adhesion to endothelial cells treated with LDLs. A, Cells were treated with 200 µg/mL LDL or vehicle control in 96-well plates and fluorescence was measured before and after labeled monocytes were allowed to adhere and nonadherent monocytes were washed out. The percent of remaining fluorescence was calculated individually for each experimental well. Absolute data varied in the ranges of 622 to 685 and 12 to 58 U for total and remaining fluorescence measurement respectively. n=4 per point, *P<0.01, **P<0.001 vs. either vehicle-treated or nLDL-treated cells, #P<0.05 vs. oxLDL-treated cells at 24 hours. B, Representative images are prepared with the cells treated in a 6-well plate. Endothelial cells are visualized with phase-contrast (gray) and labeled monocytes are detected using fluorescent microscopy. Noticeable shrinkage and decreased density of HCAECs after cLDL or oxLDL treatment. Monocytes are adherent to remaining endothelial cells. Control cells were treated with vehicle or nLDL. Scale bar, 50 µm.

Our next step experiment showed that freshly isolated human monocytes also have higher rate of adherence to endothelial cells pretreated with both cLDL and oxLDL (Figure 2A, 2B). Further, to see whether modified LDL-activated endothelial cells still attract monocytes under flow conditions, the adhesion experiments using laminar flow chambers were performed (Figure 2C, 2D). Our results demonstrate that similar to static adhesion experiments with both U937 cells and freshly isolated monocytes, HCAECs treated with modified LDLs attract more monocytes than vehicle-treated or nLDL-treated cells.


Figure 2
View larger version (41K):
[in this window]
[in a new window]

  		Figure 2. Adhesion of freshly isolated primary human monocytes to endothelial cells treated with LDLs. Adherent monocytes were visualized by microscopy (A) and quantified by measurement of total fluorescence (B). Cells were treated with 200 µg/mL LDL or vehicle control in 6-well plates, and fluorescence was measured before and after labeled monocytes were allowed to adhere and nonadherent monocytes were removed by washing. n=3 per point, *P<0.05 vs. either vehicle-treated or nLDL-treated cells. Scale bar, 25 µm. Absolute data varied in the ranges of 1100 to 1252 and 243 to 336 U for total and remaining fluorescence measurement, respectively. C and D, Adhesion of monocytes to HCAECs monolayer under flow conditions. Endothelial cells were treated with 200 µg/mL LDL or vehicle control for 16 hours after and perfused with U937 monocytes as described in Materials and Methods. n=3 per point, *P<0.05 vs. either vehicle-treated or nLDL-treated cells. Scale bar, 25 µm.

cLDL Induces ICAM-1 and VCAM-1 Expression in Endothelial Cells
We studied the expression of adhesion molecules, ICAM-1, VCAM-1, and P-selectin, and a chemokine, MCP-1, which could potentially mediate cLDL-induced monocyte adhesion to endothelial cells. HCAECs were treated with varying concentrations of cLDL or nLDL for 24 hours, and the expressions of adhesion molecules were determined using cell ELISA (Figure 3). The data showed that the expression of MCP-1 and P-selectin was not induced by the treatment with cLDL in comparison to nLDL. However, the expression of ICAM-1 and VCAM-1 was significantly increased by cLDL. ICAM-1 was induced to a higher degree then VCAM-1. Contrary to cLDL, oxLDL did not affect ICAM-1 and VCAM-1 expression, whereas expressions of P-selectin and, to a lesser extent, MCP-1 molecules were increased. The nLDL did not cause any significant change of either ICAM-1 or VCAM-1 expression.


Figure 3
View larger version (15K):
[in this window]
[in a new window]

  		Figure 3. Cell ELISA measurements of the ICAM-1, VCAM-1, P-selectin, and MCP-1 expression levels in endothelial cells after treatment with nLDL, cLDL, or oxLDL (50 to 400 µg/mL) for 24 hours. n=4 to 5 per point, *P<0.03, **P<0.01, ***P<0.001 vs. cells treated with nLDL at the same concentration. Cells treated with vehicle were considered to be a baseline (100%). The values of 100% are 0.62, 0.70, 0.89, and 0.95 optical density units for the ICAM-1, VCAM-1, P-selectin, and MCP-1, respectively.

The results of cell ELISA regarding ICAM-1 and VCAM-1 expression after cLDL treatment was confirmed by immunocytochemistry: cLDL-treated HCAECs expressed significantly more ICAM-1 and VCAM-1 in comparison to the vehicle control or nLDL-treated cells (Figure 4).


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		Figure 4. Expression of ICAM-1 and VCAM-1 mRNA in endothelial cells after cLDL treatment as measured by real-time reverse-transcription polymerase chain reaction. HCAECs were treated with 200 µg/mL cLDL for 16 hours in 6- well plates. Control cells were treated with vehicle or 200 µg/mL nLDL. Absolute C(t) data varied in the ranges of 17.2 to 17.8 cycles and 18.1 to 21.5 cycles for 18s and ICAM-1 or VCAM-1, respectively. n=3 per point, *P<0.05, **P<0.01, ***P<0.001 vs. same treatment at 0 hour; #P<0.05, ##P<0.01 vs. nLDL-treated cells at the same time point.

To determine whether the observed induction of ICAM-1 and VCAM-1 is present at the level of transcription, real-time PCR was performed with RNA extracted from the vehicle-treated, cLDL-treated (200 µg/mL), or nLDL-treated (200 µg/mL) endothelial cells. Our results suggest that the transcription of both studied molecules was upregulated at 4 and 8 hours (supplemental Figure I, available online at http://www.atvb.ahajournals.org). The mRNA expression of both molecules was decreasing at later time points.

ICAM-1 and VCAM-1 Mediate cLDL-Induced Monocyte Adhesion to Endothelial Cells
To determine whether cLDL-induced ICAM-1 and/or VCAM-1 overexpression cause the adhesion of monocytes to endothelial cells, 2 approaches were applied. In the first, we abolished the function of ICAM-1 and VCAM-1 with antibodies, and in the second, the expression of these adhesion molecules was silenced by using siRNA.

Because of the superficial location of adhesion molecules in the endothelium, the antibodies to these proteins are widely used to determine the role of adhesion molecules in endothelial cells.5,22 Our experiments showed that the inhibition of ICAM-1 caused a significant {approx}30% reduction of monocyte adhesion to endothelial cells, whereas the inhibition of VCAM-1 had only a minor and nonsignificant effect (Figure 5). In the same experiment, simultaneous pretreatment of endothelial cells with both anti–ICAM-1 and anti–VCAM-1 antibodies caused the most significant inhibition of monocyte adhesion ({approx}60%). Rabbit {gamma}-immunoglobulins used as a negative control did not have a significant effect on monocyte adhesion regardless of the treatment.


Figure 5
View larger version (21K):
[in this window]
[in a new window]

  		Figure 5. Inhibition of monocyte adhesion by antibody to ICAM-1 or VCAM-1. HCAECs were treated with 200 µg/mL cLDL for 16 hours in 96-well plates. Control cells were treated with either vehicle or 200 µg/mL nLDL. Two hours before the application of labeled monocytes, anti–ICAM-1, anti–VCAM-1, or both antibodies were added to HCAECs (final concentration of 10 ng/mL). Nonspecific IgGs served as antibody treatment control. Absolute data varied in the ranges of 784 to 801 and 24 to 67 U for total and remaining fluorescence measurement respectively. n=3 to 4 per point, *P<0.05, **P<0.01 vs. vehicle control cells pretreated with the same antibody; #P<0.05, ##P<0.001 vs. no antibody control cells (white bars) subjected to the same treatment.

Before using RNA interference, we determined the optimal conditions for siRNA transfection using siRNA-fluorescein isothiocyanate and specific siRNA, because HCAECs are notoriously difficult cells to transfect.23 These experiments showed that the optimal conditions for siRNA transfection to the endothelial cells are 60% to 70% confluence with a transfection time of no more then 48 hours. These conditions allowed reaching a transfection level of up to 70% to 80% (supplemental Figure IIA). The introduction of the specific siRNA resulted in a significant inhibition of ICAM-1 or VCAM-1 expression in modified LDL-treated cells as determined using real-time reverse-transcription polymerase chain reaction (supplemental Figure IIB).

After the application of siRNAs to the HCAECs for 48 hours, the cells were treated for an additional 16 hours with vehicle, cLDL, or nLDL and monocyte adhesion was measured as described in the Methods section. The data presented in Figure 6 show that cLDL caused accelerated monocyte adhesion to endothelial cells, and it was significantly suppressed by anti-ICAM-1 siRNA. Anti–VCAM-1 siRNA had only a partial effect while simultaneously using both anti–ICAM-1 and anti–VCAM-1 siRNAs caused the most prominent and significant suppression of monocyte adhesion. It did not reach the level of cells treated with the vehicle or nLDL, but it was consistent with the transfection efficiency observed in this experiment. Although nLDL did not accelerate monocyte adhesion, it was slightly suppressed by specific siRNAs. The control siRNA did not protect the endothelial cells from monocyte adhesion. These experiments provide evidence that ICAM-1 in cooperation with VCAM-1 is involved in monocyte adhesion by cLDL-activated human endothelial cells in vitro.


Figure 6
View larger version (58K):
[in this window]
[in a new window]

  		Figure 6. Inhibition of monocyte adhesion by siRNA to ICAM-1 or VCAM-1. A, HCAECs were transfected with anti–ICAM-1 or anti–VCAM-1 siRNA for 48 hours and then exposed with 200 µg/mL cLDL for 16 hours. Control cells were treated with vehicle or 200 µg/mL nLDL. The monocyte adhesion was measured as described in Methods. Absolute data varied in the ranges of 798 to 811 and 43 to 126 U for total and remaining fluorescence measurement, respectively. n=3 to 4 per point, *P<0.01, **P<0.001 vs. vehicle control cells pretreated with the same siRNA, #P<0.05, ##P<0.01 vs. no siRNA control cells (white bars) subjected to the same treatment. B, Representative images of cells treated with cLDL.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
This study for the first time to our knowledge determined that cLDL induces the adhesion of monocytes to human vascular endothelial cells in vitro and identified the adhesion molecules, which are important in this process. The cLDL was demonstrated to induce ICAM-1 and VCAM-1 and had no effect on MCP-1 or P-selectin. These 4 molecules and some others were described before to be involved in the monocyte adhesion to the endothelium.24 As opposed to cLDL, oxLDL, the most studied modified LDL, was shown to induce the expression of P-selectin25 and MCP-1.26 There is conflicting evidence regarding the involvement of ICAM-1 and VCAM-1 in oxLDL-induced monocyte–endothelial adhesion. While some studies showed that oxLDL may cause the induction of ICAM-1 and VCAM-1 expression and monocyte adhesion,21,27 others suggested that oxLDL does not induce VCAM-128 or either one.26,29 In support of this, Khan et al observed no change in ICAM-1 or VCAM-1 expression as measured by ELISA of cultured HAECs or human umbilical vein endothelial cells that were incubated with oxLDL or glycated LDL.30 Our data suggest that oxLDL action is not mediated by ICAM-1 or VCAM-1. Therefore, our subsequent experiments were aimed to study cause–effect relationship between cLDL-induced expression of ICAM-1 and VCAM-1 and monocyte adhesion. We have shown that unlike the other modified LDLs, cLDL induces ICAM-1 and VCAM-1 in a dose-dependent manner. The protein increase of ICAM-1 and VCAM-1 was strongly associated with the mRNA increase and monocyte adhesion. Therefore, there is a possibility that cLDL is a more potent inducer of monocyte adhesion than other modified LDLs.

Both ICAM-1 and VCAM-1 protein expressions were increased after cLDL treatment in 16 to 24 hours, whereas the mRNAs of both molecules reached a maximum at the 8-hour point. In endothelial cells treated with lipoproteins, ICAM-1 and VCAM-1 mRNAs were previously shown to be induced within 2 to 8 hours after the impact and then to be downregulated, whereas the proteins are usually induced later, at 8 to 48 hours after treatments.31,32 Our results suggested that after cLDL treatment, the VCAM-1 expression was increased to a lesser degree then the expression of ICAM-1. These data are in agreement with other studies suggesting higher reactivity of ICAM-1 after exposure to exogenous factors, including LDL.6,30,31 Monocyte recruiting is a multistep process that consists of capture, rolling, activation, adhesion, and transmigration. Although the roles of ICAM-1 and VCAM-1 are known to be mainly in adhesion and transmigration, their inactivation may in some cases also affect rolling.24 We can speculate that cLDL is involved in all of these processes.

Using cause–effect relationship approaches by using specific siRNA or neutralizing antibodies, we found that ICAM-1 and VCAM-1 are the adhesion molecules responsible for the endothelial cell attraction of monocytes induced by cLDL. Our studies also indicate that ICAM-1 is more important for cLDL-induced monocyte adhesion than VCAM-1. Both siRNA and the utilization of neutralizing antibodies determined that silencing/neutralizing of ICAM-1 alone provided inhibition of monocyte adhesion to endothelial cells. VCAM-1 contributes to this process because the silencing or neutralizing of both molecules caused the maximal effect. To the best of our knowledge, reports on the use of specific anti-ICAM-1 or anti-VCAM-1 siRNA to study monocyte adhesion to endothelial cells are not available. Our observation of the predominance of ICAM-1 is in agreement with at least one previous study. Using the pretreatment of endothelium with anti-ICAM-1 and anti-VCAM-1 antibodies in a human ex vivo experiment, Crook et al reported that ICAM-1 participates in monocyte adhesion while the role of VCAM-1 is rather secondary and dependent on the degree of its expression in the endothelium.33 It is likely that cooperation between the molecules promotes monocyte adhesion. For example, monocyte adhesion induced by a high glucose concentration was inhibited by simultaneous use of anti–ICAM-1, anti–VCAM-1, and anti–CD-18 antibodies.34 Taken together with the previous study of cLDL cytotoxicity to HCAECs in vitro,10 it can be concluded that cLDL produces a variety of atherosclerotic-prone signals to endothelial cells. Some of these signals may be different from the effects of other modified LDLs.

Several experiments in this study use high concentrations of cLDL, 50 to 400 mg/L, which may raise a question whether these conditions are physiologically relevant. While several LDL modifications have been identified, only 2 of them, oxLDL and cLDL, can be precisely measured in human plasma using sandwich ELISAs. Malondialdehyde-modified LDL, an isoform of oxLDL, was detected in healthy individuals at 1.9±0.2 µg/mL,35 3.1±1.6 µg/mL,36 and 17.1±50.2 µg/mL.37 The oxLDL concentration was 0.5±0.3 U/µg38 or 10.8±2.8 U/mL39 LDL protein (1 U was 1 µg of mildly oxLDL). The cLDL concentration determined in our recent study showed a much higher value of 86.0±29.7 µg/mL.11 It reached {approx}300 µg/mL in uremic patients with chronic renal failure, who are known to be predisposed to atherosclerosis. Therefore, the cLDL concentrations used in this study are physiologically meaningful.

Future studies may be focused on determining whether cLDL-induced adhesion results in increased rolling, arrest, and transmigration of monocytes.40 If observations from this study are confirmed in vivo, future therapeutic approaches may be aimed at the inhibition of ICAM-1 and VCAM-1 as protective measures to attenuate the progression of atherosclerosis. In this regard, using siRNA that we have tested or inhibiting the signal transduction pathways leading to induction of the adhesion molecules, for example, NF-{kappa}B–activating signaling pathways,41 would be highly appropriate.


*    Acknowledgments
 
The authors thank Ray Biondo, MD, MS, for editorial assistance, and Anna G. Stewart for technical assistance.

Sources of Funding

This research was supported by a grant from Satellite Healthcare (A.G.B., S.V.S.), VA Merit review grants (A.G.B., S.V.S.), fellowships from the Turkish Nephrology Association and the International Society of Nephrology (E.O.), and an Arkansas Tobacco Settlement Award (E.O.A.).

Disclosure

None.


*    Footnotes
 
Original received September 14, 2006; final version accepted January 5, 2007.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Postea O, Krotz F, Henger A, Keller C, Weiss N. Stereospecific and redox-sensitive increase in monocyte adhesion to endothelial cells by homocysteine. Arterioscler Thromb Vasc Biol. 2006; 26: 508–513.[Abstract/Free Full Text]
  2. Herold S, von Wulffen W, Steinmueller M, Pleschka S, Kuziel WA, Mack M, Srivastava M, Seeger W, Maus UA, Lohmeyer J. Alveolar epithelial cells direct monocyte transepithelial migration upon influenza virus infection: impact of chemokines and adhesion molecules. J Immunol. 2006; 177: 1817–1824.[Abstract/Free Full Text]
  3. Schleser S, Ringseis R, Eder K. Conjugated linoleic acids have no effect on TNFalpha-induced adhesion molecule expression, U937 monocyte adhesion, and chemokine release in human aortic endothelial cells. Atherosclerosis. 2006; 186: 337–344.[CrossRef][Medline] [Order article via Infotrieve]
  4. Chang YJ, Holtzman MJ, Chen CC. Interferon-gamma-induced epithelial ICAM-1 expression and monocyte adhesion. Involvement of protein kinase C-dependent c-Src tyrosine kinase activation pathway. J Biol Chem. 2002; 277: 7118–7126.[Abstract/Free Full Text]
  5. Silverman MD, Tumuluri RJ, Davis M, Lopez G, Rosenbaum JT, Lelkes PI. Homocysteine upregulates vascular cell adhesion molecule-1 expression in cultured human aortic endothelial cells and enhances monocyte adhesion. Arterioscler Thromb Vasc Biol. 2002; 22: 587–592.[Abstract/Free Full Text]
  6. Basnakian AG, Apostolov EO, Yin X, Abiri SO, Stewart AG, Singh AB, Shah SV. Endonuclease G promotes cell death of non-invasive human breast cancer cells. Exp Cell Res. 2006; 312: 4139–4149.[CrossRef][Medline] [Order article via Infotrieve]
  7. Hsiai TK, Cho SK, Reddy S, Hama S, Navab M, Demer LL, Honda HM, Ho CM. Pulsatile flow regulates monocyte adhesion to oxidized lipid-induced endothelial cells. Arterioscler Thromb Vasc Biol. 2001; 21: 1770–1776.[Abstract/Free Full Text]
  8. Kita T, Kume N, Ishii K, Horiuchi H, Arai H, Yokode M. Oxidized LDL and expression of monocyte adhesion molecules. Diabetes Res Clin Pract. 1999; 45: 123–126.[CrossRef][Medline] [Order article via Infotrieve]
  9. Kraus LM, Kraus AP, Jr. Carbamoylation of amino acids and proteins in uremia. Kidney Int Suppl. 2001; 78: S102–S107.[CrossRef][Medline] [Order article via Infotrieve]
  10. Ok E, Basnakian AG, Apostolov EO, Barri YM, Shah SV. Carbamylated low-density lipoprotein induces death of endothelial cells: a link to atherosclerosis in patients with kidney disease. Kidney Int. 2005; 68: 173–178.[CrossRef][Medline] [Order article via Infotrieve]
  11. Apostolov EO, Shah SV, Ok E, Basnakian AG. Quantification of carbamylated LDL in human sera by a new sandwich ELISA. Clin Chem. 2005; 51: 719–728.[Abstract/Free Full Text]
  12. O’Byrne D, Devaraj S, Islam KN, Collazo R, McDonald L, Grundy S, Jialal I. Low-density lipoprotein (LDL)-induced monocyte-endothelial cell adhesion, soluble cell adhesion molecules, and autoantibodies to oxidized-LDL in chronic renal failure patients on dialysis therapy. Metabolism. 2001; 50: 207–215.[CrossRef][Medline] [Order article via Infotrieve]
  13. Basnakian AG, Apostolov EO, Ok E, Yin X, Altunel E, Shah SV Carbamylated LDL but not oxidized LDL is associated with atherosclerosis in uremic patients. Proceedings of the XLIII ERA-EDTA Congress. July 15–18, 2006, Glasgow, UK. 2006:abstract MP400.
  14. Apostolov EO, Basnakian AG, Yin X, Ok E, Shah S Modified LDLs induce proliferation-mediated death of human vascular endothelial cells through the MAPK pathway. Am J Physiol Heart Circ Physiol. In press.
  15. Kume N, Arai H, Kawai C, Kita T. Receptors for modified low-density lipoproteins on human endothelial cells: different recognition for acetylated low-density lipoprotein and oxidized low-density lipoprotein. Biochim Biophys Acta. 1991; 1091: 63–67.[Medline] [Order article via Infotrieve]
  16. Koga T, Claycombe K, Meydani M. Homocysteine increases monocyte and T-cell adhesion to human aortic endothelial cells. Atherosclerosis. 2002; 161: 365–374.[CrossRef][Medline] [Order article via Infotrieve]
  17. Peterson MD, Jin R, Hyduk S, Duchesneau P, Cybulsky MI, Waddell TK. Monocyte adhesion to xenogeneic endothelium during laminar flow is dependent on alpha-Gal-mediated monocyte activation. J Immunol. 2005; 174: 8072–8081.[Abstract/Free Full Text]
  18. Frahm SO, Rudolph P, Dworeck C, Zott B, Heidebrecht H, Steinmann J, Neppert J, Parwaresch R. Immunoenzymatic detection of the new proliferation associated protein p100 by means of a cellular ELISA: specific detection of cells in cell cycle phases S, G2 and M. J Immunol Methods. 1999; 223: 147–153.[CrossRef][Medline] [Order article via Infotrieve]
  19. Langer I, Atassi G, Robberecht P, Resibois A. Eriochrome Black T inhibits endothelial cell growth through S-phase blockade. Eur J Pharmacol. 2000; 399: 85–90.[CrossRef][Medline] [Order article via Infotrieve]
  20. Li D, Mehta JL. Antisense to LOX-1 inhibits oxidized LDL-mediated upregulation of monocyte chemoattractant protein-1 and monocyte adhesion to human coronary artery endothelial cells. Circulation. 2000; 101: 2889–2895.[Abstract/Free Full Text]
  21. Kita T, Kume N, Minami M, Hayashida K, Murayama T, Sano H, Moriwaki H, Kataoka H, Nishi E, Horiuchi H, Arai H, Yokode M Role of oxidized LDL in atherosclerosis. Ann N Y Acad Sci. 2001; 947: 199–205.[Abstract/Free Full Text]
  22. Saijo Y, Perlaky L, Wang H, Busch H. Pharmacokinetics, tissue distribution, and stability of antisense oligodeoxynucleotide phosphorothioate ISIS 3466 in mice. Oncol Res. 1994; 6: 243–249.[Medline] [Order article via Infotrieve]
  23. Chen J, Liu Y, Liu H, Hermonat PL, Mehta JL. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) transcriptional regulation by Oct-1 in human endothelial cells: implications for atherosclerosis. Biochem J. 2006; 393: 255–265.[CrossRef][Medline] [Order article via Infotrieve]
  24. Huo Y, Ley K. Adhesion molecules and atherogenesis. Acta Physiol Scand. 2001; 173: 35–43.[CrossRef][Medline] [Order article via Infotrieve]
  25. Zhao SP, Xu DY Oxidized lipoprotein (a) enhanced the expression of P-selectin in cultured human umbilical vein endothelial cells. Thromb Res. 2000; 100: 501–510.[CrossRef][Medline] [Order article via Infotrieve]
  26. Dwivedi A, Anggard EE, Carrier MJ. Oxidized LDL-mediated monocyte adhesion to endothelial cells does not involve NFkappaB. Biochem Biophys Res Commun. 2001; 284: 239–244.[CrossRef][Medline] [Order article via Infotrieve]
  27. Erl W, Weber PC, Weber C. Monocytic cell adhesion to endothelial cells stimulated by oxidized low density lipoprotein is mediated by distinct endothelial ligands. Atherosclerosis. 1998; 136: 297–303.[CrossRef][Medline] [Order article via Infotrieve]
  28. Jeng JR, Chang CH, Shieh SM, Chiu HC. Oxidized low-density lipoprotein enhances monocyte-endothelial cell binding against shear-stress-induced detachment. Biochim Biophys Acta. 1993; 1178: 221–227.[Medline] [Order article via Infotrieve]
  29. Frostegard J, Haegerstrand A, Gidlund M, Nilsson J. Biologically modified LDL increases the adhesive properties of endothelial cells. Atherosclerosis. 1991; 90: 119–126.[CrossRef][Medline] [Order article via Infotrieve]
  30. Khan BV, Parthasarathy SS, Alexander RW, Medford RM. Modified low density lipoprotein and its constituents augment cytokine-activated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells. J Clin Invest. 1995; 95: 1262–1270.[Medline] [Order article via Infotrieve]
  31. Vielma SA, Mironova M, Ku JR, Lopes-Virella MF. Oxidized LDL further enhances expression of adhesion molecules in Chlamydophila pneumoniae-infected endothelial cells. J Lipid Res. 2004; 45: 873–880.[Abstract/Free Full Text]
  32. Lee H, Lin CI, Liao JJ, Lee YW, Yang HY, Lee CY, Hsu HY, Wu HL. Lysophospholipids increase ICAM-1 expression in HUVEC through a Gi- and NF-kappaB-dependent mechanism. Am J Physiol Cell Physiol. 2004; 287: C1657–C1666.[Abstract/Free Full Text]
  33. Crook MF, Southgate KM, Newby AC. Both ICAM-1- and VCAM-1-integrin interactions are important in mediating monocyte adhesion to human saphenous vein. J Vasc Res. 2002; 39: 221–229.[CrossRef][Medline] [Order article via Infotrieve]
  34. Manduteanu I, Voinea M, Serban G, Simionescu M. High glucose induces enhanced monocyte adhesion to valvular endothelial cells via a mechanism involving ICAM-1, VCAM-1 and CD18. Endothelium. 1999; 6: 315–324.[Medline] [Order article via Infotrieve]
  35. Holvoet P, Perez G, Zhao Z, Brouwers E, Bernar H, Collen D. Malondialdehyde-modified low density lipoproteins in patients with atherosclerotic disease. J Clin Invest. 1995; 95: 2611–2619.[Medline] [Order article via Infotrieve]
  36. Bosmans JL, Holvoet P, Dauwe SE, Ysebaert DK, Chapelle T, Jurgens A, Kovacic V, Van Marck EA, De Broe ME, Verpooten GA. Oxidative modification of low-density lipoproteins and the outcome of renal allografts at 1 1/2 years. Kidney Int. 2001; 59: 2346–2356.[Medline] [Order article via Infotrieve]
  37. Kotani K, Maekawa M, Kanno T, Kondo A, Toda N, Manabe M. Distribution of immunoreactive malondialdehyde-modified low-density lipoprotein in human serum. Biochim Biophys Acta. 1994; 1215: 121–125.[Medline] [Order article via Infotrieve]
  38. Itabe H, Yamamoto H, Imanaka T, Shimamura K, Uchiyama H, Kimura J, Sanaka T, Hata Y, Takano T. Sensitive detection of oxidatively modified low density lipoprotein using a monoclonal antibody. J Lipid Res. 1996; 37: 45–53.[Abstract]
  39. Kohno H, Sueshige N, Oguri K, Izumidate H, Masunari T, Kawamura M, Itabe H, Takano T, Hasegawa A, Nagai R. Simple and practical sandwich-type enzyme immunoassay for human oxidatively modified low density lipoprotein using antioxidized phosphatidylcholine monoclonal antibody and antihuman apolipoprotein-B antibody. Clin Biochem. 2000; 33: 243–253.[CrossRef][Medline] [Order article via Infotrieve]
  40. Devaraj S, Davis B, Simon SI, Jialal I. CRP promotes monocyte-endothelial cell adhesion via Fc{gamma} receptors in human aortic endothelial cells under static and shear flow conditions. Am J Physiol Heart Circ Physiol. 2006; 291: H1170–H1176.[Abstract/Free Full Text]
  41. Huang WC, Chan ST, Yang TL, Tzeng CC, Chen CC. Inhibition of ICAM-1 gene expression, monocyte adhesion and cancer cell invasion by targeting IKK complex: molecular and functional study of novel alpha-methylene-gamma-butyrolactone derivatives. Carcinogenesis. 2004; 25: 1925–1934.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
27/4/826    most recent
01.ATV.0000258795.75121.8av1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Apostolov, E. O.
Right arrow Articles by Basnakian, A. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Apostolov, E. O.
Right arrow Articles by Basnakian, A. G.

ATVB Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2007 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.